7. Project Summary Over the past 25 years, the prevalence of digestive diseases worldwide has increased by ~25%.1 Within the past year, intestinal organoids derived from hiPSCs have resulted in pioneering in vitro studies of colorectal cancer2, inflammatory bowel disease3, and intestinal response to infection8. Because the conversion efficiency of somatic cells into hiPSCs is low (~0.01-1%10), it is essential that the hiPSCs be purified quickly and efficiently after reprogramming to allow for rapid and successfully development of hiPSC-derived intestinal models. Current purification methods range from non-specific and time-intensive morphological analysis12 to flow cytometry sorting based on surface markers which imposes high shear forces onto hiPSCs often resulting in unwanted differentiation15. To streamline the application of hiPSCs in intestinal disease models, a universally simple, unbiased, and gentle technology for identifying and isolating hiPSCs must be developed. I aim to address the limitations of existing hiPSC sorting methods by creating an automated technology based upon microraft arrays. Microraft arrays are microwell arrays with each individual microwell containing a magnetic releasable cell culture element (?microraft?).23 Each microraft cell carrier can be gently released from the array and easily collected with a magnet for assays of their adhered cells. The proposed platform will advance microraft arrays to allow for every micro-colony of transduced erythroblasts cultured on the array to be biopsied and each colony fragment collected into a designated microwell within a replicate well array. Within the collection wells, all colony fragments are assayed simultaneously via cell-lethal immunoassays for key pluripotency biomarkers: OCT4, SOX2, TRA-1-60, and SSEA4. By mapping the assayed fragments back to the living original colony on the microraft array, the original colonies that are determined to express distinct markers of hiPSCs can be resampled, and the cells validated via genomic and differentiation assays. Studies made possible using the platform will result in new insight into the relationship between the primary hiPSC colonies? proliferation rate and biomarker expression, as well as the established hiPSC colonies? potential for self-renewal, trilineage differentiation, and development into intestinal cells. It is important to note that while this proposal limits its scope to isolating hiPSCs, the microraft array platform can easily be adapted to enrich for essentially any rare cell population with a distinct intracellular or surface biomarker, such as sub- populations of cancer cells and successfully gene-edited or differentiated cells. The platform will also provide researchers with a gentle, biomarker-based method for sorting sensitive cell types and performing higher throughput cell sorts based on intracellular markers.